of a-factor pheromone and monitored their ability
to arrest in G1 and grow a mating projection (Fig.
3A). Previous experiments that identified an age-associated mating defect used assays sensitized
with a low concentration (less than 20 ng/ml) of
a-factor (3). Indeed, we found that old mother
cells (mean age, 14.3 divisions) responded less efficiently to mating pheromone than did young cells;
however, they responded efficiently with pheromone concentrations above 20 ng/ml (Fig. 3B). If
the observed loss of mating depended on expression
from HML, then deletion of HML would have restored the sensitivity of old cells to the level of
young cells, which was not the case (Fig. 3C).
Young and old yeast cells in which HMLa2 was
deleted were more responsive to a-factor than
were wild-type cells (Fig. 3C and fig. S3). Arrest
with a-factor did not affect the stability of silencing
at HML, indicating that the heightened sensitivity
in hmla2D mutants did not reflect transcription
from HML during a-factor treatment (fig. S4).

Efficient response to mating pheromone depends on arrest in the G1 phase of the cell cycle.
Cells exposed to a-factor for longer than 4 hours
escape this cell cycle arrest and become less sensitive to pheromone (14). This adaptation depends
on aggregation and subsequent inactivation of
Whi3, an RNA-binding S-phase inhibitor, but desensitized mothers produce daughters that are
fully sensitive to a-factor (14). Old cells showed
a similar asymmetric inheritance of mating competence: The daughters of old cells were more
responsive to a-factor than were their mothers
(Fig. 4A). To test whether aggregation of Whi3
might explain why old yeast mother cells fail to
respond to a-factor, we deleted a glutamine-rich
domain required for Whi3 aggregation in cells
adapted to a-factor and assayed old MATa cells
carrying this deletion for a-factor responsiveness.
Deletion of the Whi3 glutamine-rich domain decreased the loss of sensitivity in old cells, indicating
that Whi3 aggregation may prevent mating in
old cells (Fig. 4B). Live-cell imaging of old yeast
mother cells expressing a GFP-tagged Whi3 indicated that old yeast cells did form aggregates
of Whi3 (Fig. 4, C and D, and fig. S5). Interestingly,
whi3-DpolyQ strains lived slightly longer than
wild-type strains, suggesting that aggregation
of Whi3 might limit life span (Fig. 4E).

Our conclusions regarding whether aging affects
gene silencing contrast with those of previous
work, but the methods that we used were more
sensitive and extensive than those available in
the past. Although it would be best to repeat
analyses with exactly the same strains used previously, those strains have been lost over the
years, precluding a direct comparison. Nevertheless,
our data establish that age-dependent loss of gene
silencing is not a feature of widely used budding
yeast strains.

The mechanism by which HML-deleted yeastcells have slightly decreased sensitivity to matingpheromone, independent of transcription at thelocus, is unclear. Interruption of the silent HMLlocus could have an indirect effect on mating-factor sensitivity, perhaps by inducing changes inthe three-dimensional architecture of chromo-some III that affect expression of genes involvedin the a-factor response.Both yeast and vertebrates are rich in RNA-binding proteins containing low-complexity prion-like domains. Unlike aggregates of typical yeastprions, Whi3 aggregates are sequestered in themother cell during cell division (14). Aggregationduring aging may be an intrinsic liability foryeast memory factors (mnemons) such as Whi3that encode memory in the form of protein ag-gregates. In this view, aging-induced aggrega-tion of Whi3 would preclude a-factor–inducedWhi3 aggregation as a memory of past unsuc-cessful mating encounters. It is tempting tospeculate that in nature, yeast could benefitfrom a bona fide differentiation between oldand young cells, with some aggregates beingbeneficial and others not. Understanding whyWhi3 aggregates form and are retained in themother cell during mitosis may shed light onhow protein aggregation influences the mitoticinheritance of cytoplasmic factors more broadly.

We thank M. Delarue, N. Azgui, and the University of
California–Berkeley biotechnology nanofabrication facility for
assistance in fabricating microfluidic devices; E. Unal and G. Brar for
microscopy support; S. Guetg for providing the hml::GFP reporter;
S. S. Lee for advice on microfluidics; the light microscopy center of
ETH Zürich (ScopeM); T. Schwarz for technical support; and
T. Kruitwagen for critical reading of the manuscript. RNA sequencing
data from Ellahi et al. (11) are available from the National Center
for Biotechnology Information (NCBI) under accession numbers
SRX884375, SRX885291, SRX885292, SRX885297, SRX885304, and
SRX885305, and RNA sequencing data from Sen et al. (12) are
available from NCBI as series GSE65767. This work was supported
by a grant from the National Institutes of Health (GM31105 to G.S.
and J.R.), ETH Zürich and the European Research Council project
BarrAge (to Y.B.), and an iPHD fellowship from SystemsX.ch (to M.R.K.).

The ability of light to carry and deliver orbital angular momentum (OAM) in the form
of optical vortices has attracted much interest. The physical properties of light with
a helical wavefront can be confined onto two-dimensional surfaces with subwavelength
dimensions in the form of plasmonic vortices, opening avenues for thus far unknown
light-matter interactions. Because of their extreme rotational velocity, the ultrafast
dynamics of such vortices remained unexplored. Here we show the detailed spatiotemporal
evolution of nanovortices using time-resolved two-photon photoemission electron
microscopy. We observe both long- and short-range plasmonic vortices confined to deep
subwavelength dimensions on the scale of 100 nanometers with nanometer spatial
resolution and subfemtosecond time-step resolution. Finally, by measuring the angular
velocity of the vortex, we directly extract the OAM magnitude of light.

Since the theoretical work of Poynting (1) in 1909 and the experiments by Beth (2, 3) from 1935–1936, it has been known that light can carry angular momentum. For almost a century, it was entirely attrib-uted to the coordinate-independent “spin” an-gular momentum (±ħ per photon, where ħ isPlanck’s constant h divided by 2p), associatedwith right- and left-circularly polarized light. Fol-lowing the pioneering work by Allen et al. (4), itwas realized that light can also carry orbital an-gular momentum (OAM) in the circulating Poyn-ting flow associated with helical wavefronts of anoptical vortex field. A number of demonstrations